The corticoids are a particular type of steroid having the basic carbon skeletal formula: ##STR1## which contains 21 carbon atoms in 4 rings, A thru D.
The A-D rings of the steroid nucleus being relatively planar will have some groups, at C-11 for instance, which are positioned above (.beta.) the plane of cyclopentenophenanthrene nucleus and are designated by --R and others which are positioned below (.alpha.) the plane and are designated by R.
A well-known example of the corticoids is hydrocortisone or cortisol which is represented by formula II: ##STR2##
The pharmaceutical utility of the corticoids is well known to those skilled in the art. They are used for relief of inflammatory manifestations, endocrine disorders, adrenocortical insuffieiency, rheumatic disorders, dermatologic diseases, allergic states, ophthalmic diseases, respiratory diseases, hematologic disorders, neoplastic diseases, edematous states, etc.
The corticoids are administered orally, topically or parenterally in dosages and at intervals which are well known to those skilled in the art.
Many of the most potent corticoids are characterized by having a double bond between carbon atoms one and 2. This double bond between carbon atoms one and 2 is designated by .DELTA..sup.1. Examples of some of the more common .DELTA..sup.1 -corticoids are prednisone (III), methylprednisolone (IV), triamcinolone (V), dexamethasone (VI), betamethasone 17.alpha.-valerate (VII), and fluocinolone acetonide (VIII). ##STR3##
A new topical anti-inflammatory corticoid which is very potent both with and without occlusion is diflorasone diacetate (IX). See Belgium Pat. No. 796,560, British Specification 1,403,962, and German Offen. 2,308,731. The chemical formula of diflorasone diacetate is ##STR4##
Chemical methods of introducing the .DELTA..sup.1 double bond into steroids are known. See J. Fried and J. A. Edwards, Reactions in Steroid Chemistry, Van Nostrand Reinhold Co. (1972), Vol. 1, Chapter 6. Some methods include the use of DDQ, chloranil and selenium dioxide, see, C. Djerassi, Steroid Reactions, Holden-Day Inc., San Francisco, 1963, page 231-4. Also used are dimethylacetamide and DMF-lithium carbonate.
Microbial fermentation methods for introducing the .DELTA..sup.1 double bond into steroids are also well known. See, A. Capek et al., Microbial Transformations of Steroids, Academia, Prague, 1966, page 29-30; Microbial Transformation of Steroids and Alkaloids, H. Ilzuka and A. Naito, University of Tokyo Press, Tokyo, 1967, pages 115-138 and Microbial Transformations of Steroids, W. Charney and H. L. Herzog, Academic Press, New York, 1967, page 236. In fact the microbial method of introducing the .DELTA..sup.1 double bond into the steroid nucleus is a cleaner reaction yielding a less complex mixture of products. Hence, the microbial method of .DELTA..sup.1 dehydrogenating a steroid is clearly superior to the chemical method and is the method used by the pharmaceutical industry today. For example, see U.S. Pat. Nos. 2,776,927, 2,776,928, 2,793,164, 2,822,318, 2,902,410, 2,902,411, 2,922,973, 2,951,016, 2,957,893, 2,958,631, 2,962,512, 2,968,595, 2,993,839, 3,009,937, 3,037,912, 3,037,914, 3,037,915, 3,084,103, and 3,087,864.
However, it was realized very early that .DELTA..sup.1 dehydrogenation of corticoids and pregnanes by most .DELTA..sup.1 dehydrogenating fungi resulted in destruction of the C-17 side chain. During .DELTA..sup.1 dehydrogenation by most fungi the C-17 side chain is degraded with the resulting .DELTA..sup.1 steroidal product having a C-17 alcohol, C-17 ketone or the D ring rearranged to form a 6 membered lactone. See Capek, supra, page 29; Charney, supra, pages 7-8 and U.S. Pat. No. 3,556,944. This problem is best understood by looking at a few examples:
Y. J. Abul-Hajj, J. Biol. Chem., 247, 686 (1972), and R. C. Meeks, et al., Chem. & Industry 391 (1958) reported the following reaction: ##STR5##
Also reported by Abul-Hajj, supra, was the following reaction: ##STR6##
Using a different fungus, E. Vischer and A. Wettstein, Experientia 9, 371 (1953) reported ##STR7##
M. Nishikawa, et al., Pharm. Bull. (Japan) 3,322 (1955) using the same substrate (progesterone) and fungus (F. solani) identified 2 reaction products in addition to the one reported by Vischer. They are the C-17 alcohol and the 6 member D ring lactone and are set forth below: ##STR8##
H. C. Murray and O. K. Sebek, Bact. Proc. 34 (1960), K. Singh, et al., Steroids 2, 513 (1963) and K. Singh and S. Rakhit, Biochem. Biophys. Acta 144, 139 (1967) all fermented progesterone XV with S. affinis and all obtained the formula XVI 17-keto compound and the formula XVII 17.beta.-hydroxy compound.
J. Fried, et al., JACS 75, 5764 (1953) fermented a corticoid, Compound S, with a .DELTA..sup.1 dehydrogenating fungus, Cylindrocarpon radiciola, and observed lactonization of the steroidal D ring: ##STR9##
Murray, supra, K. Singh, et al., supra, and K. Singh, et al. Can. J. Microbial. 11, 351 (1965) all reported using Compound S as a substrate, but used S. affinis instead of C. radiciola and all obtained the formula XVI and XVII compounds.
Therefore, from the above examples it is readily apparent that if one starts with a pregnane or cortical steroid and subjects it to most .DELTA..sup.1 dehydrogenating fungi the products obtained do include the .DELTA..sup.1 double bond but in addition the side chain at the C-17 position is degraded. The C-17 position in the product is either a ketone, a hydroxyl group or the D ring is enlarged to 6 members forming a lactone. Thus, regardless of whether one starts with progesterone XV or Compound S XIX the resulting products are identical since the only difference in the starting material is the side chain at C-17.
Thus, even though most .DELTA..sup.1 dehydrogenating fungi are useful in synthesizing the .DELTA..sup.1 double bond to form the highly active .DELTA..sup.1,4 A-ring they cannot always be used for this process because of the destruction of the side chain at C-17.
In fact the C-17 side chain cleavage of 20-ketopregnanes by vegetative cells of Septomyxa, and other fungi, is the subject of U.S. Pat. No. 3,556,944 (Miller). The patented process utilizes the C-17 side chain degradation to produce 17-ketoandrostanes from 20-ketopregnanes while inhibiting D-ring lactonization.
There have been reports of introduction of a .DELTA..sup.1 double bond into corticoids and pregnanes by some .DELTA..sup.1 dehydrogenating fungi without degradation of the C-17 side chain. See, for example, Iizuka, supra, p. 119, 122, 125, 127, 129-131 and 133.
Upon a close look at most of the papers reporting side chain retention during .DELTA..sup.1 dehydrogenation by a fungus, it is noted that indeed the side chain is retained - but only transiently. In fact, the reaction sequence has been so well worked out the steps are known. The steps are first, introduction of the .DELTA..sup.1 double bond, second, cleavage of the C-17 side chain to the C-17 secondary alcohol, third, oxidation to the C-17 keto compound and fourth, formation of the 6 member D-ring lactone. See, G. E. Peterson et al., J. Bacteriol. 74, 684 (1957) and M. Nishikawa Pharm. Bull. (Japan) 3,322 (1955).
M. Shiraska and M. Tsuruta, Chem. Pharm. Bull. Japan 9, 207 (1961) reported the .DELTA..sup.1 dehydrogenation of Compound S and cortisone without side chain cleavage by fungi. The fungi they used were Gliocladium roseum, Helminthosporium turcicum and Ophiobolus heterostropus. They did not use any fungi which fall within the scope of the present invention which is limited to Septomyxa.
U.S. Pat. No. 2,951,016 (Charney) discloses .DELTA..sup.1 dehydrogenation of various corticoids without side chain degradation by various molds. However, none of the organisms disclosed by Charney fall within the scope of the present invention which is limited to the genius Septomyxa. Charney disclosed that it was desirable to maintain a pH level in the reaction medium of between 6.8 and 7.2. He added, that the use of the inorganic salts for buffering the reaction mixture could be omitted. The omission of the inorganic salts causes the pH to rise from an initial value of about 6.8 to about 7.7-8.0. He stated, "this, however, will still permit the formation of the desired steroidal end products." That is not the case for the present invention. If the pH of the fermentation medium of the genus Septomyxa is not controlled so that the pH does exceed 7.0 degradation of the steroidal side chain at C-17 takes place.
G. S. Fonken et al., J. Org. Chem. 27, 1102 (1962) reported the introduction of a .DELTA..sup.1 double bond into a steroid without degradation of the C-17 side chain by S. affinis However, the steroid was a pregnane and not a corticoid which is the subject of the present invention. It is interesting to note that the author stated, "Unfortunately incubation of 5.beta.-pregnane-3,11,20-trione with S. affinis results in degradation of the side chain as well as dehydrogenation of the A ring, resulting in 5.beta.-androst-1-ene-3,11,17-trione." To prevent side chain degradation, the 20-keto group was protected by forming a 20-ethylene ketal. Upon introduction of the .DELTA..sup.1 double bond, the product was hydrolyzed to obtain the desired .DELTA..sup.1 progesterone derivative.
U.S. Pat. No. 3,770,586 (Kominek) discloses a process for .DELTA..sup.1 dehydrogenation of certain 4,9(11)-pregnadienes by S. affinis without degradation of the C-17 side chain. The steroids disclosed by Kominek are of the pregnane series and not of the corticoid type. The present invention is limited to corticoids.
S. affinis has been reported to introduce the .DELTA..sup.1 double bond into a corticoid without total side chain degradation. However, the degradation of the C-17 side chain was sufficiently great that it required two steps to resynthesize the corticoid C-17 side chain. See G. B. Spero, JACS 78, 6213 (1956).
S. affinis conidia (spores) has been reported to introduce the .DELTA..sup.1 double bond in corticoids without degradation of the side chain. See K. Singh et al., Steroids 2, 513 (1963). Conidia cannot be used on a commercial scale and are not included in the present invention.
U.S. Pat. No. 2,902,410 (Weintraub) discloses a process for .DELTA..sup.1 dehydrogenation of 20-oxygenated steroids by Septomyxa with and without degradation of the C-17 side chain. Weintraub makes no attempt to try and prevent side chain degradation when it occurs. In fact Weintraub discloses that it is an object of the invention to provide a method for the degradation of the C-17 side chain of steroids, especially the 20-oxygenated steroids by Septomyxa. Consistent with this, is the statement in column 1, lines 50-53 that a further object of the invention was a process for the production of 17-hydroxysteroids and 17-ketosteroids.